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. 2026 Feb 24;196(2):167–176. doi: 10.1007/s00360-026-01659-y

Orexinergic responses to environmental stressors in the zebrafish gut

Yingning Sang 1, Cameron T Dixon 1,2, Christina L Graves 1,✉
PMCID: PMC13233992  PMID: 41733623

Abstract

The hypothalamic hypocretin (hcrt)/orexinergic (OX) system is known to modulate sleep/wake cycles, arousal, and feeding. While orexinergic circuits have been well-described in the central nervous system, further research is needed to delineate whether gut-associated hcrt/OX signaling represents a conserved feature of vertebrate physiology. The goal of this study was to determine the presence and localization of hypocretin and its receptor in the zebrafish intestine and examine the impact of feeding state and exposure to chronic early life stress. A multimodal visualization and characterization approach was utilized which included a combination of in situ hybridization, quantitative real-time PCR, histological techniques and reporter lines. Expression was observed in the gut mucosa with morphological and anatomic evidence suggesting that epithelial cells are a major source of gut orexin in zebrafish. Feeding state was shown to modulate hcrt abundance and localization within mucosal enterocytes as well as along the vertical base-to-tip axis of the intestinal villar ridges. Gut hcrt expression appears to be positively correlated with the time of feeding. Moreover, exposure to chronic early life stress was found to negatively correlate with gut orexin expression. Altogether our data suggests that suggest gut hypocretin transcription is environmentally responsive with potential implications for the maintenance of physiological homeostasis.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00360-026-01659-y.

Keywords: Early life stress, Gut, Hypocretin, Orexin, Zebrafish

Introduction

Hypocretin/orexin as a conserved integrator of metabolic state and stress

The central hypocretin (hcrt)/orexin (OX) neuropeptide system is a highly conserved regulator of organismal homeostasis that integrates arousal, metabolic state and stress responses. Across vertebrates, hcrt/OX signaling orchestrates diverse physiological functions including sleep-wake regulation, energy homeostasis and feeding behavior, reward processing, and responses to stress (Villano et al. 2022; Kiyashchenko et al. 2002; Kukkonen et al. 2002; Dyachuk 2024). Central hcrt/OX functions are owed to a relatively small number of well-characterized hypocretin-containing neurons in the lateral hypothalamus; this central bias has shaped much of the orexin literature to date, often reinforcing the view that hcrt/OX signaling is predominantly a CNS-restricted system, despite emerging evidence for broader peripheral functions. While expression of hcrt and its receptor(s) has been reported in peripheral tissues, characterization efforts exhibit variability leading to an incomplete understanding of the presence and functional relevance of peripheral hcrt/OX orexinergic across species; here, further research is needed to delineate whether gut-associated hcrt/OX signaling represents a conserved feature of vertebrate physiology.

Comparative studies in teleosts provide important insight into the evolutionary conservation and diversification of hypocretin signaling. In zebrafish, the orexin system regulates sleep consolidation via an excitatory hypothalamic–pineal circuit that influences melatonin production, mirroring core features of mammalian orexin biology (Prober et al. 2006; Appelbaum et al. 2009; Dyachuk 2024). Notably, zebrafish possess a single functional hypocretin receptor (hcrtr), which is structurally and functionally homologous to mammalian hcrtr2, providing a simplified system for interrogating hypocretin signaling dynamics (Kaslin et al. 2004; Panula 2010; Yokogawa et al. 2007). Overall, the zebrafish model system has been described as an ideal model for interrogating central hcrt/OX networks due to the overall evolutionary conservation of the hcrt system (Dyachuk 2024).

Hcrt/OX in the Gastrointestinal tract

Mounting evidence indicates that the gastrointestinal tract is a compelling candidate site to interrogate peripheral hcrt/OX expression and function. Beyond its canonical functions in digestion, the gut is now recognized as a dynamic sensory and endocrine interface that integrates luminal nutrients, microbial metabolites, and host physiology(Rawls et al. 2004; Koh et al. 2016; Gribble and Reimann 2016). Landmark comparative studies in mammals demonstrated that hcrt/Ox expression is regionally specific throughout the gastrointestinal tract with hypocretin-like immunoreactivity reported in enteric neurons and in enteroendocrine cells supporting a role for gastrointestinal hcrt/OX in regulating food intake and digestive processes with evidence for fasting-induced activation (Kirchgessner and Liu 1999; de Miguel and Burrell 2002).

The spatial distribution and cellular localization of enteric hcrt/OX in aquatic vertebrates appears to be distinct to that of mammals, with greater variability reported between species, limiting our understanding of hcrt/OX function in the teleost gastrointestinal tract. D’Angelo et al. (2016) demonstrated that orexin A and B peptides and the OX2R receptor are expressed throughout the gastrointestinal tract of sea bass and goldfish, with immunoreactivity localized to enteric neurons and mucosal regions, supporting a conserved endocrine and peripheral role for orexin signaling in the regulation of food intake and digestive processes in teleosts (D’Angelo et al., 2016). Although hypothalamic hcrt/OX is well known to respond to feeding state and fasting in mammals (Lu et al. 2000; Sahu 2002; Diano et al. 2003; Gao and Horvath 2016), the regulation of intestinal hypocretin signaling by nutritional status in teleosts has received comparatively little attention.

Hcrt/OX in the stress response

Orexin neurons in the lateral hypothalamus and corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus (PVN) interact to modulate the hypothalamic–pituitary–adrenal (HPA) axis activity and stress modulation in mammals with data demonstrating heightened and sex-specific differential hcrt/OX responses to acute stressors (Grafe et al. 2017a, b; Grafe and Bhatnagar 2020; Moreno et al. 2005; Kuru et al. 2000). In addition, hcrt/OX signaling has been implicated in stress-associated neuropsychiatric disease (Kobayashi et al. 2022; Prajapati and Krishnamurthy 2021; Salehabadi et al. 2020). Despite clear translational relevance, the interactions between chronic stress and peripheral hcrt remain limited and whether zebrafish can be leveraged to explore these interactions has not been systematically examined.

Rationale and central hypotheses

Based on emerging evidence that hcrt/OX signaling integrates metabolic state and stress responses, and building on recent work from our laboratory demonstrating that chronic stress elicits distinct, tissue-specific neuroimmune programs in the developing zebrafish gut with lasting functional consequences (James et al. 2017; Norloff et al. 2025; Graves et al. 2023) we tested the hypothesis that fasting induces region-specific upregulation of hypocretin signaling within the zebrafish gut and that chronic stress suppresses hypocretin as an adaptive metabolic signal in both the brain and gut. To address these questions, we employed complimentary direct-detection approaches methods including quantitative real-time PCR, in situ hybridization, and histological analyses to systematically investigate whether zebrafish exhibit regionalized enteric hcrt and hcrt-receptor (hcrtr) expression, whether it localizes to epithelial versus non-epithelial compartments, and how transcription is modulated by environmental changes such as feeding state and stress exposure. Together, this work provides new insight into the spatial organization and physiological modulation of hcrt transcription in the zebrafish model system and expands our understanding of its potential role in gut–brain integration of metabolic and stress signals.

Materials and methods

Animal subjects

Procedures performed in this study were reviewed and approved by the University of North Carolina Chapel Hill (UNC-CH) Animal Care and Use Committee (protocols #20–241 and #23–178). Zebrafish were reared and maintained in the AAALAC-accredited UNC-CH Zebrafish Aquaculture Core Facility under a 14 h light/10 h dark cycle at 28 °C, pH = 7.3–7.7, and a density of 5 fish/L in compliance with the Guide for the Care and Use of Laboratory Animals as detailed on protocols.io (10.17504/protocols.io.bg3jjykn). All fish were reared on Gemma Micro 300 (Skretting USA, Tooele, UT, USA) and Ziegler’s Adult Zebrafish feed (Zeigler, Gardners, PA, USA). A total of n = 31 wildtype AB and transgenic zebrafish expressing green fluorescent protein under the hypocretin promoter region (hcrt:GFP ) were used in this study. Experimental and control groups, group size, developmental stage and sex are detailed for each methodology and/or experiment and reported in the corresponding text in each Methods subsection and/or associated figure legend as appropriate for clarity. Gut and brain tissue extracts from a subset of zebrafish exposed to a chronic early life stress (ELS) paradigm as previously published (Graves et al. 2023) were also used. To avoid learning and habituation, zebrafish were exposed to randomly selected environmental stressors comprising of strobe light flashes (10 min), tank turbulence (3 min), or chasing (5 min) administered twice per day at random during the light cycle from 6 to 30 days post fertilization (dpf) prior to euthanasia at 31 dpf. Euthanasia was performed using tricaine immersion (500 mg/L, ≥ 30 min).

RNA extraction and qRT-PCR

Adult fish of both sexes (~ 3.2 cm SL; n = 5/group for qRT-PCR and n = 6/group for RNAScope) were fasted overnight and compared to fish that received a morning feeding. Following euthanasia, whole guts were dissected and cleaned with ice-cold Dubelco’s PBS (Cat. No. 14190144; Thermo Fisher Scientific, Waltham, MA, USA) to remove contents. For regional comparisons (Fig. 1), gut samples were cut in half prior to mincing, separating the gut into proximal (S1-3) and distal (S4-7) regions of equal length using the functional regional organization system as described by Wang et al. (Wang et al. 2010). Freshly processed gut tissue was immediately homogenized using QIAshredder columns (Qiagen, Germantown, MD, USA) prior to RNA extraction using the RNeasy® Mini Kit according to manufacturer’s instructions (Qiagen). RNA quantity and purity was analyzed using a NanoDrop spectrophotometer (NanoDrop One C, Thermo Fischer Scientific, Waltham, MA, USA). cDNA synthesis was accomplished with iScript® Reverse Transcriptase (Bio-Rad Laboratories, Berkeley, CA, USA) with a starting RNA concentration of 250 ng per sample. Brain (n = 6 total with n = 3/group) and gut (n = 10 total with n = 5/group) cDNA samples derived from juvenile (31 dpf) fish comparing stressed vs. unstressed clutch mates from our previously published study (Graves et al. 2023) were also used in this study. qPCR was conducted using SsoAdvanced™ SYBR Green Supermix and Danio rerio-specific PrimePCR™ SYBR® Green Assays (Bio-Rad Laboratories, Cat. No. 10042976) specific for hcrt (Bio-Rad Unique Assay ID: qDreCDD0006267) and hcrtr2 (Bio-Rad Unique Assay IDs: qDreCED0007188, qDreCED0006267) in a 20 µl total reaction volume according to manufacturer recommendations. Briefly, 1 µl of cDNA was used as template in a two-step reaction (60 °C Tm) for 40 cycles. Data were collected and processed using CFX Connect™ and CFX Manager™ (Bio-Rad Laboratories). Gene expression was normalized using the ΔCT algorithm and compared to a reference gene (ef1α). Results are presented as normalized relative expression and fold change, as indicated in figure legends.

Fig. 1.

Fig. 1

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Transcriptional levels of hcrt and hcrtr2 in the zebrafish gut are modulated by feeding state. A hcrt and B hcrtr2 normalized expression relative to a housekeeping gene (ef1α) in the proximal (blue) and distal (orange) gut of fasted (striped) and fed (solid) adult zebrafish. C Fold change expression of hcrt and hcrt2 comparing fasted vs. fed zebrafish in the proximal and distal gut. Each dot represents one animal. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Sidak’s multiple comparisons test

Immunohistochemistry

Following euthanasia, gut tissues were cleaned in ice-cold Dubelcco’s PBS (Cat. 14190144; Thermo Fisher Scientific) to remove intestinal contents without disrupting tissue integrity. Tissues were immersed in a 10% neutral buffered formalin fixative solution (72 h, 4 °C with rocking; Cat. No. HT501128; Sigma-Aldrich, Burlington, MA, USA) prior to paraffin embedding and sectioning on a Leica RM2235 microtome (Leica Biosystems, Nussloch, Germany). Resulting 5 μm tissue sections were arranged on positively charged glass slides (Cat. No. MCOMM/W/90; StatLab Medical Products, McKinney, TX, USA) prior to baking at 60 °C for 1.5 h, xylene bath deparaffinization and rehydration using a graduated ethanol series. Heat-induced epitope retrieval was performed (120/90) using a pH = 6 citrate buffer (Cat. No. TA-135-HBL; Thermo Fisher Scientific) after which endogenous peroxidase activity was quenched using an aqueous 3% hydrogen peroxide solution. Blocking buffer (3% bovine serum albumin in PBS) was applied (1 h, RT) prior to overnight incubation (4 °C) with biotinylated rabbit α-GFP antibody (1:2000; Cat. No. 300-065-245; Lot 168232; Jackson ImmunoResearch Laboratories; West Grove, PA, USA). The VECTASTAIN Elite ABC kit was used according to manufacturer-supplied protocols and at a 1:50 dilution for signal amplification (Cat. No. PK-6100; Vector Laboratories, Newark, CA, USA). Chromogenic reaction was performed using Immpact™ VIP Peroxidase (HRP) Substrate Kit (Cat. No. SK-4605; Vector Laboratories). Sections were dehydrated in a graded ethanol series, xylene-cleared, and mounted using DPX mountant (Cat. No. 13512; Electron Microscopy Sciences, Hatfield, PA, USA) with a glass coverslip prior to imaging as described below.

RNAscope in situ hybridization

Whole guts (n = 11 total) were dissected from hcrt: egfp zebrafish, carefully cleaned to remove gut contents, and fixed in neutral buffered formalin (4 °C, 72 h). Subsequent steps were performed according to ACD Bio (Newark, CA, USA) using RNAscope® 2.5 Reagent Kit and kit manufacturer’s protocol Formalin-Fixed Parrafin-Embedded (FFPE) Sample Preparation and Pretreatment (ACD Bio document number 322452) and RNAscope® 2.5 HD Duplex Detection Kit (Chromogenic) User Manual PART 2 (ACD Bio document number 322500-USM). The target retrieval (RNAScope™ Probe-Dr-hcrt, ACD Bio Cat. No. 887231) was done using the manual method described for 5 min. For the protease step, RNAScope® Protease Plus was applied for 15 min. Brightfield images were acquired on a SLIDEVIEW™ VS200 research slide scanner (Evident Scientific, Waltham, MA, USA) using a color camera under 20X (UPlanXApo 20x/0.8 OFN26.5) and 40X objective (UPanXApo 40x/0.95 OFN26.5) magnification. Resulting images were visualized using QuPath Software (Version 0.5.0)(Bankhead et al. 2017). For the quantification, probe-positive puncta per villar (mucosa) or muscularis area were counted and calculated. A minimum of 3 independent regions per mucosa or muscularis areas in each section per sample were quantified.

Statistical analyses

Statistical analyses and graphical representations were performed using GraphPad Prism® V10.2.2 (GraphPad, La Jolla, CA, USA). The number of experimental and control subjects are shown in each figure and detailed in each associated legend. Student’s t-test was used to compare differences between two groups, one-way ANOVA was used to compare differences between > 2 groups, and statistical significance was determined at a p≤0.05 threshold unless otherwise indicated in corresponding figure legends. Data are presented as mean Inline graphic standard error of the mean unless otherwise specified.                          

Results

Acute fasting increases gut Hcrt and Hcrtr2 expression

To determine whether the proximal and distal gut of zebrafish expresses similar levels of hcrt and hcrtr2 as well as to determine whether fasting modulates gut expression of hcrt and hcrtr2, qRT-PCR was performed on gut tissue following an overnight fast (< 24 h) and compared to tankmates receiving a morning feeding. Transcriptional expression of hcrt was observed with significantly lower abundance in the proximal half of the gut (S1-S3) compared to the distal half (S4-S7); hcrt expression was increased in the fasted group with the most pronounced upregulation of hcrt observed in the distal gut of fasted fish (Fig. 1A). A similar pattern of expression was observed for hcrtr2 (Fig. 1B). Fasting induced a greater than 2-fold change (FC) in the expression of hcrt (FC: 2.4) and hcrtr2 (FC: 2.5) in the proximal gut; in the distal gut, hcrt and hcrtr2 expression increased by 8.3- and 10.0-fold, respectively (Fig. 1C).

Chronic early life stress blunts transcription of Hcrt and Hcrtr2 in the brain and gut

To determine whether the brain and gut express similar levels of hcrt and hcrtr2 as well as to determine whether chronic early life stress (ELS) modulated either expression of hcrt and hcrtr2, qRT-PCR was performed on brain and gut tissue from stress and control (unstressed) groups. Expression of hcrt was observed in both tissues, with lower overall abundance in the gut compared to the brain (Fig. 2A). hcrt expression was decreased in the stress group with the most pronounced downregulation of hcrt observed in the gut of stressed fish (Fig. 2A, C). A similar pattern of expression was observed for hcrtr2 (Fig. 2B, C). Transcription of hcrt and hcrtr2 was significantly lower in the stress group than in the control group for gut tissues; a similar trend was observed for brain (Fig. 2A-C). Specifically, we found that ELS reduced the expression of hcrt (FC: 0.35) and hcrtr2 (FC: 0.31) by over a half in brain; in gut, hcrt and hcrtr2 expression decreased by 0.24- and 0.22-fold, respectively (Fig. 2C). These results indicate that chronic ELS reduces hcrt and hcrtr2 expression in both the brain and gut of zebrafish.

Fig. 2.

Fig. 2

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Chronic ELS reduces hcrt and hcrtr2 expression in the zebrafish brain and gut. A hcrt and B hcrtr2 expression in the brain and gut of juvenile zebrafish in the absence (grey bars) or presence (green bars) of chronic early life stress. C Fold change expression of hcrt and hcrt2 in brain and gut tissues comparing stress vs. control. Each dot represents one animal. *p < 0.05; Mann-Whitney U test

Anti-GFP staining of Hcrt:eGFP gut tissue confirms robust gut Hcrt expression

To validate our findings of transcriptional expression of hcrt in the zebrafish gut, we leveraged the Tg(hcrt:GFP) reporter line which expresses GFP under the hcrt promoter and used an antibody-mediated labeling approach to further confirm the presence and location of hcrt in the gut and visceral tissues. In fed animals, we observed faint-yet-diffuse mucosal expression which appeared to be localized to the basolateral mucosal epithelium in the proximal, middle, and distal gut (Fig. 3A-C’). In the fasted group, an overall higher density of signal was observed throughout the length of the gut, consistent with our qRT-PCR data (Fig. 3D-F’). Additionally, we found that in fasted animals signal intensity was more often localized to the apical (lumen-facing) mucosa (Fig. 3E’, F’). We did not observe overt staining in the muscularis or villar core (lacteal) regions suggesting that processed full-length orexin is primarily expressed in the mucosal region of the zebrafish gut. Although inter-individual variability was observed, expression overall appeared highest in the mid and distal gut regions – areas that participate in nutrient absorption (Supp Fig. 1). Overall, these results demonstrate that fasting increases hcrt expression in the apical epithelium of the gut.

Fig. 3.

Fig. 3

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Anti-GFP Staining of hcrt:GFP gut tissue. A-C’ shows gut hcrt:GFP expression in fed fish. D-F’ shows gut hcrt expression in fasted fish for the proximal (A-A’, D-D’), mid (B-B’; E-E’) and distal (C-C’; F-F’) gut. GFP- controls are shown in G and G’. hcrt:GFP+ samples without addition of the primary anti-GFP antibody (Ab, refered to as secondary Ab only controls) are shown in H and H’. Purple = GFP+  expression; teal = nuclei staining. Scale bar = 20 μm. Images shown are representative of n = 5–6 individuals per group

RNAScope validation

RNAScope in situ hybridization was the performed on whole gut sections from adult zebrafish to validate, visualize and delineate the regional distribution of the hcrt molecular signature observed. Intriguingly, we observed hcrt RNA to be diffuse and widespread in the intestinal mucosa along the length of the zebrafish gut (Fig. 4A). We also observed diffuse detection and quantification of single RNA molecules, providing spatial gene expression results. Here, fasting significantly increased hcrt expression, with the highest expression found in the S4-5 region, although the comparison was not significant. We also observed minimal signal in the muscularis or villar core regions, similar to our immunostaining findings (Fig. 4B). Taken together, these data suggest that orexin expression in the gut is highly responsive to feeding state.

Fig. 4.

Fig. 4

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RNAScope quantification of mucosal vs. muscularis hcrt expression. A Mucosal and B muscularis regions. Proximal = S1-S3 region; Mid=S4-S5 region; Distal=S6-S7 region. Data shown are cumulative of n = 5–6 individuals per group and n = 2–6 quantitated villar regions per individual expressed as points (RNAScope positive) per area (µm2). Each dot represents one quantitated region. Brown-Forsythe and Welch ANOVA Tests with Dunnet’s T3 Multiple Comparisons test were used to determine statistical significance with a significance threshold of p ≤ 0.05

Discussion

Transcriptional modulation of gut Hcrt and Hcrtr2 in response to feeding state

Orexins are known to play a critical role in central metabolism and food intake and are activated during fasting in mammals (Blais et al. 2017; Gao and Horvath 2016; Sahu 2002; Diano et al. 2003; Lu et al. 2000). In line with this, hcrtr2 has been recently shown to modulate feeding state, supporting a conserved role for hcrtr2 in appetite regulation in zebrafish (Lewandowski et al. 2025). In tilapia, another teleost species, fish subjected to prolonged starvation showed an increase in orexin immunoreactivity in the hypothalamus and pituitary gland, overall supporting a highly conserved role for hcrt/OX in central regulation of metabolism (Gouda and Ganesh 2025). In this study, we provide evidence of an enteric hcrt system in zebrafish that is responsive to feeding state (Fig. 1). An overnight fast increased transcription of hcrt and its receptor hcrtr2 in both the proximal and distal gut; increased transcriptional signal was observed in the distal gut relative to the proximal gut both at steady state and upon fasting, where hcrt and hcrtr2 increased by about 8-fold in the distal gut compared to about a 2-fold induction in fasted animals indicating the the distal gut is more responsive to feeding state than the proximal gut (Fig. 1). In zebrafish, the distal gut represents a major site of nutrient sensing and microbial interaction indicating gut hcrt may play roles in nutrient absorption rather than other processes such as chemical digestion (Rawls et al. 2004; Bates et al. 2006).

Transcriptional modulation of gut and brain hcrt and hcrtr2 in response to chronic stress

Chronic and early-life stress has profound impacts on psychological and physiological states. We recently reported effects of chronic ELS on the gut using the zebrafish model system, which showed altered the neuroimmune profile of both the brain and gut with functional consequences to gut function (Graves et al. 2023; Norloff et al. 2025). The role of the central hypocretin system in stress responses has been recently reviewed (Sargin 2019). Previous studies have shown that acute stress increases orexin expression (Grafe and Bhatnagar 2018, 2020; Holmes 2017).More recent studies in mice indicate that chronic unpredictable mild stress suppresses the activity of orexin neurons in the lateral hypothalamus (Liu et al. 2025). Mounting experimental evidence indicates that central orexinergic circuits exhibit dynamic changes in response to environmental stressors suggesting that these pathways can be functionally modulated over short time scales (Yaeger et al. 2025). In this study, we show that exposure to a chronic stress paradigm suppressed hcrt and hcrtr2 transcription in both the gut and brain (Fig. 2) suggesting that orexin may represent one mechanism by which stress interferes with metabolic flexibility and gut–brain communication in zebrafish. Taken together these data indicate that chronic environmental stress may blunt both central and peripheral orexinergic responses, with potential implications for stress-associated growth disturbances, energy balance, and disease susceptibility (Kivimaki et al. 2023).

Differential Hcrt and Hcrtr2 expression in brain and gut tissue

As expected, hcrt and hcrtr2 expression was highest in brain tissue, with transcript levels exceeding those detected in the gut by approximately 26-fold, independent of feeding state or introduction of chronic stress, consistent with the possibility that the CNS remains the primary locus of hypocretin signaling (Fig. 2). At the same time, many gut hormones, such as glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are known to act at picomolar concentrations that are orders of magnitude lower levels than that of centrally expressed neuropeptides while still exerting potent physiological effects through high-affinity receptors (Holst 2007; Batterham et al. 2002). Taken together, the dynamic regulation of hcrt/hcrtr2 observed in the gut indicates functional relevance locally albeit at levels much lower than that of the central nervous system. Notably, the hcrtr2:hcrt expression ratio remained remarkably consistent across tissues and experimental conditions (~ 1.2–1.4), an invariance consistent with tight, homeostatic regulation similar to what has been overserved with other neuropeptides and gut hormone systems including GLP-1 (Muller et al. 2019).

Epithelial localization and regionalization of Hcrt in the zebrafish gut

Immunohistochemical staining of gut tissue derived from hcrt:GFP zebrafish revealed anti-GFP staining that appeared largely restricted to the mucosal epithelium, with a marked absence of staining in the submucosal/lamina propria and muscularis regions (Fig. 3, Supp Fig. 1). A similar intratissue expression pattern with low muscularis signal was also observed using RNAscope RNA in situ hybridization strengthening interpretation of our findings (Fig. 4). The epithelial-restricted staining and low submucosal/lamina propria and muscularis signal are consistent with the hypothesis that gut hypocretin is locally regulated and not simply a byproduct of generalized tissue expression or due to nonspecific staining. Intriguingly, we also observed strong apical enrichment in fasted fish indicating that hcrt may play a role in luminal nutrient sensing, intestinal secretion, digestive mechanisms, or mediating interactions with host microbiota, although these hypotheses were not tested in the present study. Given that orexinergic neurons in the hypothalamus directly sense blood glucose (Viskaitis et al. 2024; Venner et al. 2011), future studies should explore whether hcrt in absorptive enterocytes play an analogous role in the periphery, potentially by monitoring glucose derived from intestinal gluconeogenesis and functioning as a local epithelial neuroendocrine signal through autocrine or paracrine mechanisms. In line with this possibility, the short chain fatty acid, acetate, has been shown to modulate orexin/hypocretin neurons suggesting that enteric microbial metabolites can regulate orexin activity via the gut-brain axis in mice (Forte et al. 2024). Additionally, microbiota-derived acetate can indirectly stimulate intestinal gluconeogenesis, thereby linking luminal microbial metabolism to epithelial glucose production and central metabolic sensing (De Vadder et al. 2014). Whether local or epithelial hypocretin signaling acts as an intermediate linking microbial metabolite–induced intestinal gluconeogenesis to central metabolic circuits remains an open and testable question.

Conceptual model

We propose a model in which central and intestinal hcrt represent coordinated-yet-separable arms of integrated metabolic state and stress responses. Within this framework, central hcrt is hypothesized to primarily coordinate systemic adaptations (e.g. arousal, activity, feeding behavior), whereas gut hcrt is hypothesized to function locally as a nutritional-state sensor and epithelial effector. A third component of this model is that chronic stress may dampen both arms, reducing organismal capacity to mount appropriate adaptive responses to energetic challenge. Importantly, this model is intended to guide future directions rather than serve as definitive conclusion; follow up studies should determine temporal aspects of brain vs. gut hcrt responses, mapping hcrt expression to defined epithelial cell types in zebrafish and explore how chronic stress modulates fasting-induced engagement of hcrt.

Limitations and future directions

This study was designed to interrogate tissue-specific patterns of hypocretin expression under fasting and chronic stress conditions and therefore does not address all potential cellular or functional mechanisms underlying these observations; this work largely focused on transcriptional responses, an important caveat given that full-length orexin is proteolytically processed into peptidergic constituents limiting our ability to directly test peripheral orexinergic activity with functional assays. Future studies should interrogate cell-type specificity with particular attention to the absorptive epithelium and enteroendocrine lineage cells. Additionally, we utilized tissue cross sectioning to enable visualization of intratissue architecture and the gut epithelial, mucosal, submucosal and muscularis regions simultaneously. At the same time, sectioning techniques can limit the 3D-spatial resolution of the muscularis externa, potentially occluding signals arising from enteric neuron bodies or processes; here, ex vivo whole-mount gut tissue preparations may be required to fully resolve whether the lack of muscularis signal observed in this study is due exclusively to non-neuronal expression. Overall, our work establishes zebrafish as a model to interrogate tissue-specific orexin biology and suggest that epithelial cells may play underappreciated roles in peripheral orexinergic regulation in mammalian systems.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (2.3MB, docx)

Acknowledgements

The authors would like to thank Michelle Altemara and all the staff at the Zebrafish Aquaculture Core for zebrafish husbandry, Wendy Salmon from the UNC Hooker Imaging Core Facility for microscopy and imaging, and Ashley Ezell and all the staff at the UNC Histology Research Core Facility for histological services. The Tg(hcrt:GFP) line used in this study was a kind gift of Dr. David Prober. The authors would also like to thank Heidi Lindborg, Adam Lietzan, and all members of the Graves Laboratory for thoughtful discussion.

Author contributions

CG conceived and designed the research & interpreted results of experiments. CG and YS performed experiments and analyzed data and prepared figures. CG, CD, and YS drafted, edited, and revised the manuscript. All authors approved the final manuscript.

Funding

This research was supported by a NIAID Ruth L. Kirschstein National Research Service Award (NRSA) Institutional Research T32 Training Grant [T32-AI007273; R. Tisch] appointment (to C.G.), a Pilot Award from NC TraCS, Grant Number: 2KR1432103 (to C.G.), as well as start-up funds from the Adams School of Dentistry (to C.G.). The research was additionally supported by a 2023 Summer Undergraduate Research Fellowship from the UNC-Chapel Hill Office for Undergraduate Research (to Y.S.) as well as a UNC Senior Honors Thesis Research Award (to Y.S.). The UNC Hooker Imaging Core Facility is supported in part through a Cancer Center Core Support Grant with Grant Number: NIH P30 CA016086 (to the UNC Lineberger Comprehensive Cancer Center; Boucher). This work was also supported in part by an Institutional Research and Academic Career Development Award (IRACDA) from the National Institute of General Medical Sciences (NIGMS) grant #K12GM000678 (C.D.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Data availability

All data generated or analyzed during this study are available from the corresponding author upon request.

Declarations

Conflict of interest

The authors disclose no conflict of interest. The authors declare no disclaimers.

Footnotes

Publisher’s note

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (2.3MB, docx)

Data Availability Statement

All data generated or analyzed during this study are available from the corresponding author upon request.

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